Specific binding reactions, such as antibody-antigen interactions, have been used extensively in immunoassays to detect and/or quantify numerous analytes present in biological fluids. Immunoassays have been developed for the determination of polyvalent antigens as well as haptens.
Of particular interest for clinical diagnostic purposes are haptens belonging to various classes of drugs, including drugs of abuse. Examples of such drugs of abuse include amphetamines, barbiturates, benzodiazepams, cocaine, methadone, methaqualone, opiates, phencyclidine (PCP), propoxyphene, tetrahydro-cannabinol (THC), and compounds of related structures.
The preparation of antibodies useful in immunoassays for the determination of such haptens is complicated in that these relatively small molecules can act as epitopes but, by themselves, are generally incapable of eliciting an immune response. Thus, in order to obtain antibodies specific for particular haptens of interest it is often necessary to immunize an appropriate host animal with an immunogenic conjugate of the hapten of interest or a derivative thereof and a carrier. Various known carriers include poly(amino) acids such as proteins and polypeptides. Commonly employed carriers include keyhole limpit hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, and pumpkin seed globulin.
Immunogenic conjugates useful in eliciting antibodies against haptens, including haptens having an amine functionality, have been prepared by modifying or derivatizing a hapten of interest to contain a reactive group, such as a carboxyl group, to facilitate its binding with a functional group, such as an amine group, of an immunogenic carrier substance such as a protein.
French Patent No. 2561660, having a publication date of Mar. 26, 1984, issued to Geffard, discloses an immunogen against a hapten group which contains a choline unit, prepared by reacting an amine or carboxyl group of an antigenic molecule with a compound of the formula Y--X.sub.2 --CO--Z', where X.sub.2 is an alkyl group, Z' is either an OH group or protective group, and Y is a functional group capable of reacting with an amine or carboxyl group of a macromolecule, followed by esterification of the resulting coupled compound with choline.
U.S. Pat. No. 4,490,473 issued to Brunhouse on Dec. 25, 1984, discloses N,N,N-trimethylammonium phenyl compounds conjugated to an antibody through an amino linkage of the antibody. These compounds are disclosed as reagents useful in sandwich assays.
U.S. Pat. No. 4,078,049 issued to Felix et al. on Mar. 7, 1978, discloses an antigen comprising 6-aminocaproic acid (2-trimethylaminoethyl) ester covalently bonded to an immunogenic carrier material through a peptide bond formed from the 6-amino group and carboxyl groups contained in the immunogenic carrier.
European Patent Appln. 0191536, Visser et al., having a publication date of Aug. 20, 1986 discloses immunogens comprising an antigenic determinant coupled to an amphiphilic adjuvant molecule, and optionally, free amphiphilic adjuvant molecules.
U.S. Pat. No. 3,709,868 issued to Spector, on Jan. 1, 1973, discloses the coupling of an opium alkaloid hapten to immunogenic carrier proteins through the carboxyl group of a carboxy lower alkyl derivative of the phenolic hydroxy group of the alkaloid with a free amino group in the protein, thereby yielding a covalent peptide bond to form an opium alkaloid antigen.
None of these references disclose a quaternary ammonium immunogenic conjugate prepared by coupling a hapten or derivative thereof to an immunogenic carrier by attaching a bifunctional linking group capable of binding to the carrier, to a tertiary amine group present in the hapten or derivative thereof.
Haptens or derivatives thereof can also be conjugated with non-immunogenic carriers such as reporters to provide reporter reagents useful in immunoassays. Conjugates of haptens and a solid phase, such as latex particles, can provide latex particle reagents which can be used in various known particle agglutination immunoassay formats. For example, U.S. Pat. No. 4,401,765 issued Aug. 30, 1983 to Craig et al. and U.S. Pat. No. 4,480,042 issued Oct. 30, 1984 to Craig et al. disclose high refractive index shell core polymer particle reagents comprising biological compounds of interest coupled to particles and the use of these particle reagents in light scattering particle agglutination or inhibition immunoassays.
Quaternary ammonium compounds can be prepared from tertiary amines using known methodology involving alkylation of the nitrogen atom. Alkylation can generally be achieved through reaction with an alkyl halide. The amine can be aliphatic or aromatic. Such quaternary ammonium compounds have the general formula, [R.sub.4 N.sup.+ ]X.sup.-, where R.sub.4 represents four non-hydrogen substituent groups and X is a halide anion. See for example, Morrison R. T. and Boyd, R. N., Organic Chemistry, sixth edition, page 854, Prentice Hall Inc. (1992) .
However, quaternary ammonium reporter reagents which are conjugates of reporters and reporters or derivatives thereof which have a tertiary amine functionality have not been previously disclosed.